AIDS Kit I: Simulation of HIV-1 Detection - Frederiksen

EDVOTEK • P.O. Box 1232 • West Bethesda, MD 20827-1232 

® 

The Biotechnology 

Education Company ® 

AIDS Kit I: 

Simulation of 

HIV-1 Detection 

271 

EDVO-Kit # 

Storage: 

Store entire experiment in the refrigerator. 

Experiment Objective: 

The objective of this experiment is to understand the molecular biology 

of the human immunodeficiency virus and the pathogenesis of acquired 

immune deficiency syndrome. The experimental concepts and methodology 

involved with enzyme linked immunosorbent (ELISA) assays will 

be introduced in the context of the clinical screening of serum samples 

for antibodies to the HIV virus. 

1-800-EDVOTEK • (301) 251-5990 • 24-hour FAX: (301) 340-0582 

http://www.edvotek.com • email: edvotek@aol.com 

All components are intended for educational research only. They are not to be used for 

diagnostic or drug purposes, nor administered to or consumed by humans or animals. 

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EDVO-Kit # 271: AIDS Kit I: Simulation of HIV-1 Detection 

EDVOTEK • The Biotechnology 


Major Section Headings 

Page 

Experiment Components 3 

Requirements 3 

Background Information 4 

Experimental Procedures 9 

Study Questions 12 

Instructor's Guide 

General Information 13 

Pre-Lab Preparations 13 

Avoiding Common Pitfalls 16 

Idealized Schematic of Results 16 

Answers to Study Questions 17 

This experiment does not contain HIV virus or its components. 

None of the components have been prepared from human sources. 

Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. This 

document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. 

Copyright © 1992,1993,1994,1996,1997,1998, EDVOTEK, Inc., all rights reserved. 

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Experiment Components 

Storage: 

Store entire experiment 

in the refrigerator. 

All components of this experiment 

are intended for educational 

research only. They are not to be 

used for diagnostic or drug 

purposes, nor administered to or 

consumed by humans or animals. 

This kit is designed for 10 lab groups 

A 

B 

C 

D 

E 

F 

G 

H 

Contents 

HIV Antigens (simulated) 

Positive Control (primary antibody) 

Donor 1 Serum (simulated) 

Donor 2 Serum (simulated) 

Anti-IgG-peroxidase conjugate (secondary antibody) 

Hydrogen peroxide, stabilized 

Aminosalicylic acid (peroxide co-substrate) 

Phosphate buffered saline concentrate 

2 Microtiter plates 

50 Transfer pipets 

60 Microtest tubes with attached caps 

25 1 ml pipets 

2 plastic tubes, 50 ml 

This experiment does not contain HIV virus or its components. 

None of the components have been prepared from human sources. 

Requirements 

• Distilled or deionized water 

• Beakers 

• 37°C Incubation oven 

• Disposable lab gloves 

• Safety goggles 

• Automatic micropipets and tips recommended 

Make sure glassware is clean, dry and free of soap residue. 

For convenience, additional disposable transfer pipets (Cat. # 632) can 

be purchased for liquid removal and washing steps. 




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BACKGROUND INFORMATION 

Background Information 

Acquired immune deficiency syndrome (AIDS) is a disease characterized 

by the progressive deterioration of an individual's immune system. The 

immunological impairment allows infectious agents such as viruses, bacteria, 

fungi and parasites to invade the body and propagate unchecked. In 

addition, the incidence of certain cancers dramatically increases in these 

patients because of their compromised immune system. AIDS is a serious 

threat to human health and is a global problem. Intensive research is being 

done to advance methods of detection, clinical treatment and prevention. 

Retroviruses 

The AIDS etiologic agent (HIV-1) is the human immunodeficiency virus 

type 1, which is a retrovirus. Retroviruses contain an RNA genome and 

the RNA dependent DNA polymerase also termed reverse transcriptase. 

Members of the retrovirus family are involved in the pathogenesis of certain 

types of leukemias and other sarcomas in humans and animals. The 

structure and replication mechanism of HIV is very similar to other 

retroviruses. HIV is unique in some of its properties since it specifically 

targets the immune system, is very immunoevasive, forms significant 

amounts of progeny virus in vivo during the later stages of the disease and 

can be transmitted during sexual activity. 

Glycoprotein 

Reverse 

transcriptase 

Protease 

The HIV viral particle is surrounded by a lipid bilayer derived from the 

host cell membrane during budding. The viral proteins are identified by 

the prefix gp (glycoprotein) or p (protein) followed 

Protein 

coat 

RNA 

by a number indicating the approximate molecular 

weight in kilodaltons. The lipid bilayer contains gp 

160, gp 120 and gp 41. The gp 41 anchors gp 120 in 

the bilayer. Beneath the bilayer is a capsid consisting 

of p17 and p18. Within this shell is the viral core. 

The walls of the core consists of p24 and p25. Within 

the core are two identical RNA molecules 9000 nucleotides 

in length. Hydrogen bonded to each genomic 

RNA is a cellular tRNA molecule. The core also contains 

approximately 50 molecules of reverse transcriptase. 

Lipid 

bilayer 

Core 

Integrase 

Large quantities of virus can be grown in tissue culture 

for diagnostic and research purposes. Several 

of the viral proteins have been cloned and expressed 

in relatively large quantities. 




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Background Information, 

continued 

Mechanism of HIV Infection 

An individual can be infected with HIV through an abrasion in a mucosal 

surface (e.g. vaginal and rectal walls), a blood transfusion or by 

intravenous injection with a contaminated needle. Virus or virally infected 

cells are found in bodily fluids such as semen and blood. During 

the early stages of infection in an immunocompetent person the 

HIV virus elicits humoral and cellular immunity responses that result 

in a variety of circulating IgG molecules directed at several viral 

epitopes. However, since the virus has a high mutation rate the variants 

survive and produce progeny having a similar capacity to escape 

immunosurveillance. 

Unlike other cellular DNA polymerases, HIV DNA polymerase (reverse 

transcriptase) has a high error rate (1 in 10 4 ). These frequent 

mutations continually change the viral protein epitopes. This is believed 

to be the main mechanism of HIV immunoevasion. The most 

important target for the virus are hematopoietic cells such as bone 

marrow derived monocytes, myelocytes and immune system lymphocytes. 

Infection of immune system effector cells such as T cells and 

macrophages ultimately produce the most profound clinical consequences. 

gp 120 binds to the CD4 receptors on the surface of T helper 

(T H 

) cells. These receptors are membrane bound glycoproteins involved 

in T cell maturation from precursor cells. T H 

cells are required for the 

body's overall immunological responses. The viral lipid bilayer fuses 

with that of the cell's membranes and the viral protein capsid becomes 

internalized via receptor mediated endocytosis. Subsequently, the rest 

of the CD4 receptors are internalized and gp 120 appears on the T cell 

surface. 

HIV Replication and Transcription 

Through a complex mechanism involving several events, the reverse 

transcriptase synthesizes a double stranded DNA copy of the genomic 

RNA template. The tRNA molecule acts as the primer for the first 

strand synthesis. The reverse transcriptase, RNAse H activity, degrades 

the RNA strand of the RNA-DNA duplex and the polymerase activity 

synthesizes a complementary DNA strand. The DNA reverse transcripts 

(copy DNA) migrate into the cell nucleus where they become 

covalently integrated into the cellular genomic DNA. The integration 

is catalyzed by the HIV integrase. The copy DNA integrates via specific, 

self-complimentary sequences at both ends called long terminal 

repeats (LTRs). These sequences also have important functions in viral 

transcription. The integrated copy DNA is called proviral DNA or 

the provirus. The provirus enters a period of latency that can last for 

several years. The proviral DNA is replicated along with the cellular 

DNA and can be inherited through many generations. The HIV proviral 

DNA contains the major genes common to all non-transducing 




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continued 

retroviruses. These genes are gag, pol and env. HIV also contains five 

or six other genes that are much smaller. Retroviral transcription is a 

complex process producing a variety of RNAs. Promotion of transcripts 

is controlled in the LTR and transcriptional termination signals are located 

in each major gene. Those RNA transcripts that remain unspliced 

become packaged in the new viral particles. The gag gene is translated 

into a polypeptide that is cleaved by a viral protease into four 

proteins that form the inner shells. Specific protease inhibitors are clinically 

being used to inhibit protein processing and control the further 

spread of the HIV virus in patients suffering from AIDS. The pol gene 

encodes the reverse transcriptase and the integrase which is responsible 

for the genomic incorporation of copy DNA. The env gene encodes 

the surface glycoproteins the viral particles acquire as they bud 

from the cells. 

Immunological Response: 

Macrophages are circulating monocytes and are involved in the nonspecific 

engulfment of foreign material and normal cellular debris. 

These materials are degraded in the lysosomes of the cells. Peptides 

from foreign degraded proteins are transported to the macrophage 

surface where they remain bound by specialized receptors. Immunologically 

inactive T H 

cells interact with these surface bound antigenreceptor 

complexes which enables them to become fully activated. HIV 

infects macrophages by binding to the cells' CD4 receptors. Fully activated 

T H 

cells secrete several types of protein factors collectively known 

as lymphokines. Several of these factors are the interleukins which 

stimulate antibody secretions from B cells enable macrophage activation, 

stimulate general T cell growth and activate cytotoxic T cells. 

Cytotoxic T cells are involved in the actual destruction of foreign cells 

and body cells infected with different viruses. Inactive T H 

cells that 

have been infected by HIV remain in a relatively quiescent state similar 

to uninfected cells. When a T H 

cell containing provirus undergoes 

antigenic activation the integrated copy DNA becomes open to the transcription 

of viral RNA. 

Viral replication causes the destruction of the T H 

cells. Infected T H 

cells 

also form syncytia, i.e. fused cells. Syncytia occur since the gp 120 on 

the infected T cell surface binds to CD4 receptors on other T H 

cells. 

Cell to cell transmission of virus can occur in syncytia without the need 

for a free viral intermediate. Replication of virus also proceeds in activated 

macrophages which causes cell death and release of infectious 

viral particles. These and other events ultimately cause complete immune 

system collapse. The long latency period after HIV infection can 

be understood in terms of T H 

cell activation. Only certain T H 

cells are 

capable of responding to a particular antigen. HIV infected but 

asymptomatic individuals will experience the usual exposure to chemicals, 

viruses and bacteria. Each infection activates a subpopulation T H 

cells containing the provirus which eventually leads to the death of 




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continued 

these cells. After successive waves of infections the population of T H 

and macrophage cells become depleted and clinical AIDS develops. 

There are a very small number of individuals who have coexisted with 

HIV for over 15 years. Although the reasons for this coexistence with 

HIV is not understood, the immune system appears to remain intact. 

Such individuals tend to eat well, exercise and practice stress reduction 

techniques. Genetic analysis may determine whether or not subtle 

genetic differences in the immune system are significant factors. 

Description of the HIV Screening Simulation 

well 

Human 

Serum 

(IgG) 

HIV 

Antigen 

well 

Schematic for 

ELISA 

Horseradish 

peroxidase 

Anti-IgG 

(Rabbit) 

Human 

Serum 

(IgG) 


Antigen 

Enzyme linked immunosorbent assay (ELISA) tests were originally 

developed for antibody measurement. These immunoassays have also 

been adapted to successfully detect samples that contain antigens. This 

ELISA simulation experiment has been designed to detect a hypothetical 

patient's circulating IgG directed towards the viral (HIV) antigen. 

ELISAs are done in microtiter plates which are generally made of polystyrene 

or polyvinyl chloride. The plates are somewhat transparent 

and contain many small wells, in which liquid samples are deposited. 

First, the antigens are added to the wells 

where some remain adsorbed by hydrophobic 

association to the walls after washing 

away the excess. The antigens can be 

the whole HIV lysate, specific HIV proteins, 

or a mixture of the two. There is no 

specificity involved with the adsorption 

process although some substances may exhibit 

low binding to the walls. In certain 

cases the antigens can be covalently crosslinked 

to the plastic using UV light. After 

S 

(colorless) 

well 

Horseradish 

peroxidase 

Anti-IgG 

(Rabbit) 

Human 

Serum 

(IgG) 


Antigen 

P 

(color) 

washing away unadsorbed material, the 

unoccupied sites on the walls of the plastic 

wells are blocked with proteins, typically 

gelatin or bovine serum albumin. 

Infection by HIV-1 causes the individual 

to mount an antibody response which 

eventually results in plasma IgG molecules 

that bind to different HIV proteins (and/ 

or different areas or the same polypeptide). 

In this experiment, if these antibodies are 

present in the plasma sample they will 

bind to the adsorbed antigens in the well 

and remain there after washing. 

A solution containing the IgG antibody 

that binds to any kind of human IgG, is 




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continued 

then added to the wells. If the primary antibody has remained in a 

well, then the secondary antibody will bind to it and also remain attached 

after washing. These secondary antibodies are usually raised 

in rabbits and goats immunized with human IgG fractions. The second 

IgG antibodies are purified and covalently cross linked to horseradish 

peroxidase. This modification does not significantly affect the 

binding specificity and affinity of the antibody or the enzymatic activity 

of the peroxidase. 

After washing, a solution containing hydrogen peroxide and 

aminosalicylate is added to each well. Peroxidase possesses a high 

catalytic activity and can exceed turnover rates of 10 6 per second. Consequently, 

amplification of a positive sample can occur over several 

orders of magnitude. Many hydrogen donor co-substrates can be used 

by peroxidase. These co-substrates include o-diansidine, 

aminoantipyrine, aminosalicylic acid and numerous phenolic compounds 

that develop color upon oxidation. The substrate solution 

added is nearly colorless. Peroxidase converts the peroxide to H 2 

O + 

O 2 

using the salicylate as the hydrogen donor. The oxidized salicylate 

is brown and can be easily observed in wells containing anti-HIV-1 

IgG (positive plasma). 

It should be noted that polyclonal antibody preparations to a given 

antigen can have variable binding affinities due to differences in the 

immunological responses between animals. Different immunizations 

with the same antigen in the same animal can also produce variable 

binding affinities. The use of monoclonal antibodies directed against a 

single epitope eliminates this variability. Western blot analysis of positive 

samples are used to confirm infection by HIV. 




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EXPERIMENTAL PROCEDURES 

EXPERIMENT OBJECTIVE: 

The objective of this experiment is to understand the molecular biology 

of the human immunodeficiency virus and the pathogenesis of 

acquired immune deficiency syndrome. The experimental concepts 

and methodology involved with enzyme linked immunosorbent 

(ELISA) assays will be introduced in the context of the clinical screening 

of serum samples for antibodies to the virus. 

LABORATORY SAFETY 

Gloves and goggles should be worn routinely as good laboratory practice. 

Student Experimental Procedures 

GENERAL INSTRUCTIONS AND PROCEDURES 

Row 1 

Row 2 

Row 3 

Row 4 

Remember! 

Equilibrate a 37°C 

incubation oven before 

starting the experiment. 

Labeling the Microtiter Plate: 

• Place the microtiter plate vertically as shown in Figure 2. 

• Mark the plate with your initials or lab group number and 

number the rows 1-4 down the side. 

Labeling the Plastic Transfer pipets: 

Label 5 transfer pipets as follows: 

• ( - ) (negative) 

• (+ ) (positive) 

• DS 1 (Donor Serum 1) 

• DS 2 (Donor Serum 2) 

• PBS (Phosphate Buffered Saline) 

Use the appropriately labeled plastic transfer pipet for liquid removals 

and washes as outlined in the experimental procedures starting on 

page 10. 

Figure 2 




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Student Experimental 

Procedures, continued 

INSTRUCTIONS FOR ADDING LIQUIDS AND 

WASHING WELLS 

Adding Reagents to wells: 

• For adding reagents to the wells, use the same 1 ml pipet. 

Useful Hint! 

The general 

procedure for adding 

reagents to the wells 

will conserve the use of 

a large number of pipets in this 

simulated classroom ELISA. 

If available, reagents should be 

dispensed with an automatic 

micropipet using disposable tips. 

• RINSE THE PIPET THOROUGHLY with distilled water before 

using the pipet for adding the next reagent. 

Liquid Removal and Washes: 

• When instructed in the experimental procedures, remove liquids 

with the appropriately labeled transfer pipet, and then 

wash the wells as follows: 

A. Use the transfer pipet labeled "PBS", to add PBS buffer to 

the wells in all rows. Add PBS buffer until each well is 

almost full. 

The capacity of each well is approximately 0.2 ml. Do not allow 

the liquids to spill over into adjacent wells. 

B. With the appropriately labeled transfer pipet, remove all 

the liquid (PBS buffer) from the wells in each row. Dispose 

the liquid in the beaker labeled "waste". 

EXPERIMENTAL STEPS FOR THE ENZYME LINKED 

IMMUNOSORBENT ASSAY (ELISA) 

1. To all 12 wells, add 0.1 ml of “HIV” (viral antigens). 

WEAR SAFETY GOGGLES 

AND GLOVES 

2. Incubate for 5 minutes at room temperature. 

3. Remove all the liquid (viral antigens) with a transfer pipet. 

4. Wash each well once with PBS buffer as described above ("Liquid 

Removal and Washes"). 

In research labs, following this step, all sites on the microtiter plate are 

saturated with a blocking solution consisting of a protein mixture, such 

as BSA. We have designed this experiment to eliminate this step to save 

time. 




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Student Experimental 

Procedures, continued 

Reminders: 

ADDING REAGENTS: 

Be sure to rinse the 1 ml pipet 

thoroughly before adding a new 

reagent. (Steps 1, 5, 9, & 13). 

Alternatively, if you are using 

automatic micropipets, use a fresh tip 

for each reagent. 

LIQUID REMOVALS: 

Use the appropriately labeled transfer 

pipet to remove all liquid from the wells 

in each row (steps 3, 7, & 11) and 

after washes (steps 4, 8 & 12) 

Transfer pipet (-) Row 1 

Transfer pipet (+) Row 2 

Transfer pipet DS 1 Row 3 

Transfer pipet DS 2 Row 4 

Dispose the liquid in the beaker 

labeled "waste". 

WASHES: 

For all rows, use the transfer pipet 

labeled "PBS" to add PBS until each 

well is almost full. (Steps 4, 8, & 12) 

5. Add reagents as outlined below: 

Remember to rinse the 1 ml pipet thoroughly with distilled water 

before adding a new reagent. If you are using automatic micropipets, 

use a clean micropipet tip for each reagent. 

• Add 0.1 ml of PBS Buffer to the three wells in Row 1. 

(This is the negative control.) 

• Add 0.1 ml of "+" (positive) to the three wells in Row 2. 

(This is the positive control.) 

• Add 0.1 ml of Donor Serum “DS1” to the 3 wells in Row 3. 

• Add 0.1 ml of Donor Serum “DS2” to the 3 wells in Row 4. 

6. Incubate at 37°C for 15 minutes . 

7. Remove all the liquid from each well with the appropriately labeled 

transfer pipet. 

8. Wash each well once with PBS buffer (as described under "Liquid 


9. Add 0.1 ml of the anti-IgG peroxidase conjugate (2°Ab) to all 12 

wells. 

10. Incubate at 37°C for 15 minutes. 

At this time you can obtain the substrate to be used in step 13. Since the 

substrate must be prepared just prior to use, your instructor will prepare 

it towards the end of the incubation in step 10. 

11. Remove all the liquid from each well with the appropriately labeled 

transfer pipet. 

Quick Reference: 

The positive control, which 

contains IgG directed against HIV 

antigens, is the primary antibody. 

Positive serum samples will also 

contain anti-HIV IgG, while 

negative serum samples will not 

contain anti-HIV IgG. 

12. Wash each well once with PBS buffer (as described under "Liquid 


13. Add 0.1 ml of the substrate to all 12 wells. 

14. Incubate at 37°C for 5 minutes. 

15. Remove the plate for analysis. 

16. If color is not fully developed after 5 minutes, incubate at 37°C for 

a longer period of time. 




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Study Questions 

1. Describe the mechanism of ELISA. Why is ELISA so sensitive 

Why is it necessary to block unoccupied binding sites in the microtiter 

wells Why is it important to have a positive control 

2. Why can the onset of AIDS take several years 

3. Why is anti-HIV-1 IgG screened instead of the virus itself 

4. Why does the destruction of T H 

cells compromise the entire immune 

system How does HIV target T H 

cells 

5. Why are there so many immunological variants of HIV 

6. The elimination of several steps in the ELISA could be accomplished 

if the primary antibody was made into an enzyme conjugate. Why 

is this generally not done What can cause a false positive in an 

ELISA 




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INSTRUCTOR'S GUIDE 

General Information 

APPROXIMATE TIME REQUIREMENTS FOR PRE- 

LAB AND EXPERIMENTAL PROCEDURES 

1. Pre-lab preparation of biologicals and reagents takes approximately 

one and one-half hours. 

2. The student experimental activity requires approximately 60 minutes. 

Pre-Lab Preparations 

PREPARATIONS BEFORE THE LAB 

Microtiter Plates 

1. As shown in Figure 1, orient the microtiter plates so that the numbers 

1-12 are at the top and the letters A-H are on your left. 

Row 1 

Row 2 

Row 3 

Row 4 

Row 1 

Row 2 

Row 3 

Row 4 

A 

B 

C 

D 

E 

F 

G 

H 

1 2 3 4 5 6 7 8 9 10 11 12 

cutting lines depicted by dashed lines 

2. Cut each plate on the dotted lines as 

shown in the figure. Each piece will be 3 

wells on one axis and 4 wells on the other 

axis. Each lab group will receive one 

piece. 

Dispensing Components A through D: 

3. Use a FRESH 1 ml pipet (provided) for 

dispensing each of the components A-D 

directly from the component tubes provided 

in this experiment kit. Label 

microtest tubes and dispense volumes as 

outlined in the chart "Quick Reference: 

Preparation of experiment reagents" 

which appears on Page 15. 

Figure 1 




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Pre-Lab Preparations, 

continued 

PREPARATIONS ON THE DAY OF THE LAB 

Preparation of Phosphate Buffered Saline 

1. Add all of the Phosphate Buffered Saline concentrate (H) to 270 ml 

of distilled water. Mix. 

2. Label this diluted Phosphate Buffered saline as “PBS”. 

3. Dispense 25 ml into small beakers for each of the 10 lab groups. 

Preparation of Anti-IgG Peroxidase Conjugate 

(Secondary Antibody) 

4. Add 0.3 ml of diluted Phosphate Buffered Saline (PBS) to the concentrated 

Anti-IgG peroxidase conjugate (E). Mix thoroughly by 

tapping and inverting the tube. 

5. Transfer 15 ml of diluted Phosphate Buffered Saline (PBS) to a 50 

ml plastic tube provided. 

6. Transfer the entire contents of tube E prepared in step 4 to the 50 

ml tube containing 15 ml of PBS. Label the tube "2°Ab" (Secondary 

Antibody). Mix. 

7. Dispense 1.4 ml of the diluted Anti-IgG peroxidase conjugate for 

each group. 

PREPARATION OF PEROXIDASE SUBSTRATE 

DURING THE LAB EXPERIMENT 

Quick Reference: 

The substrate is prepared for the 

peroxidase enzyme, which is 

attached to the anti-IgG 

peroxidase conjugate (secondary 

antibody). 

Prepare the substrate 15 - 30 

minutes before students require it 

for plate development (last 

incubation). 

Prepare 15 - 30 minutes before the last incubation: 

1. Dispense 13.5 ml of diluted Phosphate buffered saline (PBS) to the 

second 50 ml tube provided. 

2. Add Aminosalicylic acid (G) to the 13.5 ml of PBS. Cap and mix 

thoroughly by shaking and/or vortexing. There is usually undissolved 

material remaining. 

3. Then add 1.5 ml of Hydrogen peroxide (F). Cap and mix. 

4. Dispense 1.4 ml of the peroxidase substrate for each group. 




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Quick Reference Tables 

Preparation of Experiment reagents 

Dispense for 

Label each group 

A* HIV Antigens HIV 1.4 ml 

B* Positive control + 0.4 ml 

C* Donor Serum # 1 DS 1 0.4 ml 

D* Donor Serum # 2 DS 2 0.4 ml 

E + PBS Anti-IgG-peroxidase-conjugate 2°Ab 1.4 ml 

PBS + F + G Peroxidase-enzyme substrate Substrate 1.4 ml 

H + water Phosphate Buffered Saline PBS 25.0 ml 

* Components A - D can be dispensed before the actual day of the lab and stored in the 

refrigerator. If these components are dispensed on the day of the lab, leave at room 

temperature. 

STUDENT MATERIALS 

Each Lab Group Should Receive: 

1 piece of microtiter plate 

1 tube labeled "HIV" 

1 tube labeled "+" 

1 tube labeled "DS 1" 

1 tube labeled "DS 2" 

1 tube labeled "2°Ab" 

1 1 ml pipet (or 1 automatic micropipet with tips) 

1 pipet pump 

5 Transfer pipets 

1 beaker containing 16 ml of PBS 

1 beaker containing approximately 100 ml of distilled water 

1 empty beaker labeled "waste" 

1 tube labeled "Substrate" (just before the last incubation) 




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Avoiding Common Pitfalls 

If you don't find answers to your 

questions in this section, call our 

Technical Service Department 

(1-800-338-6835) 

Mon - Fri 

9:00 am to 

5:00 pm EST 

24-hour FAX: (301) 340-0582 

email: edvotek@aol.com 

1. Students should be advised to be very careful when transferring 

solutions into and out of the microliter plate wells. 

2. Use only clean or appropriately labeled pipets and avoid contaminating 

adjacent wells. 

3. Do not attempt to empty the microliter wells by shaking it out. 

This will not work - it will result in contaminating adjacent wells. 

4. Wash the wells gently and slowly, without force. 

5. As an optional activity, the plates can be read in a microplate reader 

at 405 nm. 

Please have the following 

information: 

• The kit number and title 

• Kit lot number on box or tube 

• The literature version number 

(in lower right corner) 

• Approximate purchase date 

Expected Results 

Donor 2 should show positive for HIV. 

The color should look similar to the positive 

control. 

A 

B 

1 2 3 

C 

D 




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Answers to Study Questions 

1. Adsorbed antigen is bound by an IgG. The bound IgG is itself 

bound by an IgG from another species. The second IgG is conjugated 

to a marker such as peroxidase which can produce colored 

products from certain substrates. If sites not occupied by antigens 

are not blocked, the primary and secondary antibodies will 

be non-specifically adsorbed as well, producing false positives. 

A positive control assures that the reagents and plates are working 

optimally. The sensitivity of the ELISA is due to the high 

catalytic turnover rates of the enzyme linked conjugate. One conjugate 

can generate millions of product molecules in a few minutes. 

2. HIV remains as a provirus until the T-cell is activated by a specific 

antigen. 

3. The HIV has a proviral phase. During the early stages of the disease 

very little circulating virus is present. In addition, the viral 

mutation rate is too high to dependably monitor with a given 

preparation of antibodies. 

4. T H 

cells produce lymphokines which are required for T-cytotoxic, 

B-cell and macrophage growth and maturation. The virus binds 

to T H 

cell CD4 receptors. 

5. High error rate of reverse transcriptase. 

6. For every antigen the corresponding IgG would have to be purified 

and conjugated. This is too labor intensive and not cost effective. 

False positives can be caused by the cross reactivity of an 

antibody. Occasionally, two unrelated antigens will be recognized 

by the same antibody. Inadequate blocking of the microtiter well 

will also give a false positive. 




EVT 909168AM

AIDS Kit I: Simulation of HIV-1 Detection - Frederiksen

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